There is rapidly increasing interest in developing molecular imaging approaches that enable traditional radiological imaging techniques to obtain a wide range of information about molecular and cellular processes that occur in normal and diseased tissue. A range of information is considered important such as the ability to monitor cell migration, the development of reporters that enable imaging of gene expression, the development of robust strategies to image receptors, and the development of environmentally sensitive agents that can be used to detect the presence of specific enzymes or monitor changes in ion status. The long term goals of this work are to develop strategies that enable MRI contrast that is sensitive to a wide range of molecular and cellular processes. This work builds on over 15 years of work where we have demonstrated the first MRI strategy for detecting gene expression, the first MRI approach for monitoring a surrogate of calcium influx, the first MRI approach for performing neuronal track tracing of newly born neurons, and the first MRI approach for monitoring the migration of single cells in vivo. These all represented initial reports by any radiological imaging technique which enabled these processes to be measured. These techniques are finding widespread application to imaging pre-clinical models of a broad range of diseases. Over the past year we have made progress in all of the specific aims. Aim 1: Develop iron oxide based contrast for labeling and imaging the migration of endogenous neural stem cells. Over the past few years we have demonstrated the unique advantages of micron sized iron oxide particles for MRI of specific cells. Single cells can be detected and indeed, single particles within single cells can be detected. The main paradigm for MRI of cell migration is to label cells ex vivo and monitor migration after transplantation into an animal. The ability to detect a single particle enables inefficient labeling strategies. In particular, over the past few years we have demonstrated that injection of particles into the ventricles of the rat brain enables particles to be taken up by neural precursors in the subventricular zone and MRI can monitor the migration of cells to the oflactory bulb. Over the past year we have completed a study to determine if odor deprivation affects the migration of these cells. Initial observations indicated a decrease in migration of cells. However, odor deprivation decreases volume of olfactory bulb leading to work distinguishing decrease bulb size or decreased activity leading to changes in migration of cells. Clearly the ability to get anatomy and function from the MRI has been important. We continue to extend capabilities to image the migration of single cells along the migratory pathway. A second project images the entire brain to study immune brain interactions in a model of virus infection. This work has required working out effective strategies to label T cells, a population of cell that has been very difficult to label. This offers the unique potential to follow the low level peripheral immune surveillance that occurs in the normal adult brain as well as any changes due to inflammation or degeneration. Confounds due to bleeding in the model were studied over the pas Aim 2: Apply microfabrication techniques to manufacture unique metal structures that may be valuable for MRI contrast. Iron oxide particles commonly used for MRI are very potent contrast agents enabling detection of single micron sized particles. However, due to bulk phase manufacture of particles they are not very uniform and they do not contain very high content of metal. A solution to this problem is to use modern microfabrication techniques to manufacture metal based, micron sized contrast agents. To begin this work we have explored a variety of approachs to microfabrication oof MRI contrast agents. Over the past few years we have shown that double dougnut and cylinder structures offer unique advantages for distinguishing particles. Microfabriaction of simple iron discs lead to 10 times more potent contrast than presently available particles. Over the past year we have shown that an elliptical shape also generated the unique contrast as double doughnuts and cylinders. There may be manufacturing advantages of ellipses. Aim 3: Develop novel delivery mechanisms to extend the applicability of manganese enhanced MRI. Over the past ten years we have demonstrated the remarkable utility of the manganese ion for MRI contrast. Manganese ion enters cells on ligand or voltage gated calcium channels and so can be used as an MRI agent to monitor calcium influx. Once inside of neurons, manganese will move in an anterograde direction and cross functional synapses enabling neuronal networks to be imaged with MRI. Finally, manganese given systemically gives cytoarchitectural information about the rodent brain. These successes have us interested in broadening the ways in which manganese ion can be delivered to cells. Over the past few years we have made transferrin-manganese complexes. When bound to transferrin manganese is a poor MRI contrast agent. However, when transferrin is taken up by cells it can release manganese which is then trapped intracellularly. Thus, transferrin manganese is an agent that monitors the successful endocytosis of the transferrin by its receptor. We have demonstrated the same effects with MnOxide based nanoparticles. Over the past year we have completed studies that demonstrated another approach to making Mn nanoparticles using block co-polymer synthesis. The first generation of these agents have very high relaxivities and the relaxivity can be modulated. Finally, we are completing out initial work demonstrating that manganese positron emitting isotopes will enable PET to obtain similar information that can be obtained with manganese enhanced MRI. This opens a pathway to translating our pre-clinical Mn studies to humans. Aim 4: Develop strategies that enable cellular processes to alter the relaxivity of MRI contrast agents. In specific aim 3 we demonstrated a way in which a normal biological process (endocytosis of transferrin-Mn or MnO particles) can alter the effectiveness of an MRI contrast agent. It would be very exciting to find ways in which this can occur which are sensitive to other biological processes. To this end we have begun to explore ways in which the microfabricated particles produced under Aim 2 can be modulated. Over the past year we have completed a study that demonstrated that the microfabricated particles can be made into a pH sensor . The strategy used is generalizable to sense many other processes. The block co-polymer agents also offer many possibilities for making environmentally sensitive MRI agents and over the past year we have demonstrated that they two can be made pH sensitively. Over the next year we hope to explore the advantages of these agents for targeting and being made sensitive to proteases.